Allorecognition is the ability of organisms to distinguish self from non-self, a common theme in many organisms and a central component in the evolution of multicellularity. In mammals, allorecognition is mediated by the Major Histocompatibility Complex (MHC), which is part of the immune system. The MHC facilitates identification of parasites in infection processes, and graft rejection in transplantation medicine There is evidence for the involvement of other proteins in graft rejection but there are very few model systems available to study these proteins. There are several allorecognition systems in non-mammalian systems, notably in marine organisms, but most of them are not genetically tractable. In the previous grant period we found an allorecognition system in the social soil amoeba Dictyostelium discoideum. The system is based on two proteins, TgrB1 and TgrC1 that share many properties with mammalian MHC proteins, but not their amino acid sequences. We found these trans-membrane proteins to be highly polymorphic in natural Dictyostelium populations and necessary and sufficient for allorecognition. Dictyostelium cells live as free amoebae in the soil when food is abundant but they aggregate into multicellular organisms when starved. Aggregation involves chemotaxis to extracellular cAMP and Dictyostelium is one of the best model systems for the study of chemotaxis, which is a central process in embryogenesis and in innate immunity. When Dictyostelium cells encounter cells that carry incompatible TgrB1 and TgrC1 during aggregation, they segregate from one another and form separate multicellular organisms. We propose that TgrB1 on the surface of one cell binds TgrC1 on the surface of an adjacent cell and that binding initiates a signal transduction cascade that alters cell behavior. We intend to test this hypothesis by performing allele replacement and protein-domain swapping experiments using different polymorphic alleles of the two genes. Our recent work also showed that allorecognition has an unexpected effect on chemotaxis. Many models of chemotaxis consider the process to be cell-autonomous, but our findings suggest a cooperative effect that requires interactions between cells of the same allotype. To study this revolutionary finding further, we will test the effect of allorecognition on specific aspects of chemotaxis and identify which of the well-studied chemotaxis modules is altered by allorecognition. We also propose to use transcriptome analysis and genetic suppressors to identify signal-transduction genes that integrate allorecognition. We already found several candidate genes, including chromatin remodeling components and protein kinases that are likely candidates in the process. We will study these genes in the context of allorecognition and chemotaxis and search for additional ones as well. Lastly, we propose to test the role of allorecognition in Dictyostelium development past the aggregation stage. We found that the tgrB1 and tgrC1 are enriched in prestalk cells so we propose to use conditional expression and chimeric aggregates to test the possible role of these genes in cell-type differentiation and tissue segregation.